Nodule bacteria from the cultured legume Phaseolus dumosus (belonging to the Phaseolus vulgaris cross-inoculation group) with common tropici phenotypic characteristics and symbiovar but distinctive phylogenomic position and chromid

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Abstract

Phaseolus dumosus is an endemic species from mountain tops in Mexico that was found in traditional agriculture areas in Veracruz, Mexico. P. dumosus plants were identified by ITS sequences and their nodules were collected from agricultural fields or from trap plant experiments in the laboratory. Bacteria from P. dumosus nodules were identified as belonging to the phaseoli-etli-leguminosarum (PEL) or to the tropici group by 16S rRNA gene sequences. We obtained complete closed genomes from two P. dumosus isolates CCGE531 and CCGE532 that were phylogenetically placed within the tropici group but with a distinctive phylogenomic position and low average nucleotide identity (ANI). CCGE531 and CCGE532 had common phenotypic characteristics with tropici type B rhizobial symbionts. Genome synteny analysis and ANI showed that P. dumosus isolates had different chromids and our analysis suggests that chromids have independently evolved in different lineages of the Rhizobium genus. Finally, we considered that P. dumosus and Phaseolus vulgaris plants belong to the same cross-inoculation group since they have conserved symbiotic affinites for rhizobia.

Introduction

Rhizobia are the oldest bacterial products used in agriculture and have been commercially produced for over one hundred years. Even now, biotechnology companies have a strong preference for Rhizobium strains because they are considered safe and are not potential human pathogens. Crop inoculants produced with rhizobia constitute one of the best examples of the successful use of bacteria to avoid the use of chemical fertilizers [31], which are a source of pollution [71] and represent a large cost for farmers. Additionally, the high nitrogen content of legume seeds may be related to the nitrogen input from nitrogen-fixing rhizobia [39].

Phaseolus vulgaris (common bean) is the most widely used legume for human nutrition in the world, its site of origin is the Balsas region in Mexico [7] and domestication regions have been recognized in Mesoamerica and South America [15]. P. vulgaris symbionts have been studied from many geographical regions [4], [5], [10], [12], [18], [20], [22], [30], [38], [42], [45], [46], [47], [57], [58], [61], [62], [68], [69], [70], [76], [83], [84], [86], [87]. In contrast, nodule bacteria from other Phaseolus species have been less studied, except those from other cultivated species such as Phaseolus lunatus [19], [37], [50], [51], [53], [55] and Phaseolus coccineus [76].

Among the many rhizobial species that nodulate P. vulgaris, Rhizobium tropici, Rhizobium freirei [14] and Rhizobium etli selected strains are used commercially as bean inoculants [31], [36]. Our description of R. tropici highlighted an outstanding bacterial species [44] that became the preferred inoculant for P. vulgaris in Brazil [31] and illustrates the value of rhizobial diversity studies. Several species related to R. tropici have subsequently been described commonly isolated from tropical areas [11], [13], [14], [26], [28], [63], [80] and here they are collectively referred to as the tropici group.

In Mexico, besides P. vulgaris, there are several native Phaseolus species. For example, Phaseolus albescens which is endemic and related to P. vulgaris, climbs over pine trees with vines that are over 10 m long. It is a wild non-cultivated species, and its rhizobial isolates resembled Rhizobium leguminosarum. P. albescens rhizobia had a symbiosis plasmid that was clearly related to the phaseoli plasmid (for P. vulgaris nodulation), however, it formed ineffective nodules in P. vulgaris reflecting its adaptation to P. albescens instead of to P. vulgaris [73]. Other Phaseolus with studied symbionts are Phaseolus microcarpus [74], Phaseolus acutifolius and Phaseolus spp. [56]. Phaseolus dumosus, is a high mountain endemic species whose symbionts have scarcely been studied. Mountain tops may be considered as islands in evolutionary ecology analyses. In a phylogenomic profile P. dumosus plants are closely related to Phaseolus costarricense and related to P. coccineus and P. vulgaris [60]. Previously, wild P. dumosus Rhizobium symbionts were described but were not assigned to any known species [75]. P. dumosus was among the Phaseolus species that were identified as those with a symbiont shift to form nodules with Rhizobium instead of bradyrhizobia [75].

Traditional agriculture in Mexico dates back thousands of years [24] and nowadays it is frequently found in low resource areas where farmers depend on small pieces of land. The site of study in Ocotepec, Ayahualulco, Veracruz is a marginal area of extreme poverty with traditional agricultural practices. In a previous study from Veracruz we obtained rhizobia of the tropici group from Gliricidia sepium, the alfalfa from the tropics [1], and from P. vulgaris [52] and bradyrhizobia from P. lunatus [37]. The interest in studying Phaseolus symbionts was to stimulate the cultivation of these crops, to try to motivate the inhabitants to return to traditional products that are more nutritional than the junk food that is currently consumed in Mexico, and to obtain knowledge of Phaseolus nitrogen-fixing symbionts that could help to provide regional and efficient inoculants.

Section snippets

Study site and plant collection

Plants were collected in June 2016 in the rural community of Ocotepec, Ayahualulco in the State of Veracruz, Mexico (19°21′N, 97°9′W), which is close to the region known as Cofre de Perote. The altitude is 2280 ma.s.l. with annual ranges of temperature and rainfall (in Ayahualulco) of 2–18 °C and 300–1600 mm, respectively.

The cultivation fields are distributed among the families in the community and each one takes charge of their own crop field. In order to eliminate the use of agrochemicals, the

Phaseolus plant identification

Intergenic plant ribosomal sequences (ITS) were used to identify the Phaseolus species grown by farmers in Ocotepec and revealed that cultured bean plants were P. dumosus and P. vulgaris (data not shown). It was unknown that farmers therein cultivated P. dumosus. Other cultured Phaseolus identified only by morphology with no ITS sequence determined were designated here as Phaseolus sp.

Phaseolus nodule-bacteria identification

From agricultural fields and plant-trap assays, Phaseolus nodule isolates were identified by the sequence of

Acknowledgements

To CONACYT Problemas Nacionales 246999 “Biodiversidad en la milpa y su suelo: bases para la seguridad alimentaria de mujeres y niños en el Cofre de Perote” Problemas Nacionales 246999 to Dra. Simoneta Negrete-Yankelevich and to Ciencia Básica CONACyT 253116 and PAPIIT IN207718 from UNAM to Esperanza Martínez-Romero. To Mariangela Hungria for kindly providing the unpublished genome of R. paranaense for ANI analysis.

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